Smallpox and monkeypox represent two dangerous orthopoxviruses that can cause high morbidity and mortality - as well as widespread fear in unvaccinated populations. Although the last case of natural smallpox infection occurred in 1977, it was weaponized by the former Soviet Union and remains a significant concern due to the potential that stocks of this highly contagious virus could fall into the hands of terrorists or rogue nations. In contrast to smallpox, monkeypox still causes naturally occurring outbreaks, such as the one that occurred in the United States in 2003. It is easily obtained/isolated, and has the potential to be genetically manipulated into a more lethal pathogen than it already is. Together, these viruses represent a significant threat to homeland security. Here, we present new preliminary evidence showing that we have developed a platform of diagnostic reagents that are more sensitive than the currently used PCR assay for detecting a recent monkeypox infection. In Specific Aim I of this Phase I application, we will further characterize these monkeypox-specific reagents and test the hypothesis that a similar approach will also be suitable for diagnosing smallpox infections. In Specific Aim II, we will identify "pan-pox" reagents capable of identifying any recent infection with the major orthopoxviruses known to cause human disease. Development of these reagents will serve as independent confirmation of a recent orthopoxvirus outbreak and will also provide valuable information that is relevant to future vaccine design and treatment of monkeypox or smallpox infections. It is expected that Phase II of this project will utilize the unique reagents that are created and characterized in Phase I to develop rapid, point-of-care diagnostic tests that can be used to immediately identify monkeypox or smallpox outbreaks. [unreadable] [unreadable]